Analysis of N-terminal Pyroglutamate


Analysis of N-terminal Pyroglutamate

One of common post-translational modifications in monoclonal antibodies is a cyclized N-terminal glutamine or pyroglutamic acid (PyroGlu), since either the heavy and/or light chain sequences begin with glutamine, leading to lower molecular weights and lower isoelectric points. The co-existence of N-terminal Gln and PyroGlu is a major factor contributing to antibody charge heterogeneity. Recent researches have proven that cyclization of Gln has no effect on antibody potency. A high level of PyroGlu has been reported in human endogenous IgGs, so N-terminal PyroGlu in recombinant monoclonal antibodies, currently is not considered as a drug safety concern. However, a consistent level of PyroGlu can be taken as a good indicator of a consistent robust production process.

The the presence of N-terminal PyroGlu poses a challenge for detailed characterization of charge variants because of peak overlapping. Being a source of mAb heterogeneity, the biotech companies are prior to identification of N-terminal PyroGlu and determining percentage of each variant. This analytical procedure is commonly accomplished by enzyme digestion followed by LC-MS to separate, identify and quantify the different forms. Direct determination of the cyclized N-terminal glutamine has recently been accomplished with ESI Q-TOF mass spectrometry, and our tech support can help you to make it with this instrument.

Typically, to determine the presence of N-terminal Gln or PyroGlu, antibody variants are separated by, for example, cation exchange chromatography(SXC) and collected into separate fractions, which are concentrated and analyzed by LC-MS. LC-MS measurement of the molecular weights of antibody light chain, heavy chain, and various proteolytic fragments can be used to demonstrate the presence/absence of PyroGlu by database searching. The identities of the N-terminal amino acid can ultimately be determined by LC-MS peptide mapping. Creative Proteomics have modified the protocols from reputable journals, and provide robust LC-MS based analytical service.


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